Technical Field
The present invention relates generally to compositions and methods for modulating stem cells, in particular, stem cell division symmetry, and uses thereof.
Description of the Related Art
Stem cells are undifferentiated or immature cells that are capable of giving rise to multiple specialized cell types and ultimately, to terminally differentiated cells. Most adult stem cells are lineage-restricted and are generally referred to by their tissue origin. Unlike any other cells, stem cells are able to renew themselves such that a virtually endless supply of mature cell types can be generated when needed over the lifetime of an organism. Due to this capacity for self-renewal, stem cells are therapeutically useful for the formation, regeneration, repair and maintenance of tissues.
It has recently been determined that satellite cells represent a heterogeneous population composed of stem cells and small mononuclear progenitor cells found in mature muscle tissue (Kuang et al., 2007). Satellite cells in adult skeletal muscle are located in small depressions between the sarcolemma of their host myofibers and the basal lamina. Satellite cells are involved in the normal growth of muscle, as well as the regeneration of injured or diseased tissue. In undamaged muscle, the majority of satellite cells are quiescent, meaning they neither differentiate nor undergo cell division. Satellite cells express a number of distinctive genetic markers, including the paired-box transcription factor Pax7, which plays a central regulatory role in satellite cell function and survival (Kuang et al., 2006; Seale et al., 2000). Pax7 can thus be used as a marker of satellite cells.
Upon damage, such as physical trauma or strain, repeated exercise, or in disease, satellite cells become activated, proliferate and give rise to a population of transient amplifying progenitors, which are myogenic precursors cells (myoblasts) expressing myogenic regulatory factors (MRF), such as MyoD and Myf5. In the course of the regeneration process, myoblasts undergo multiple rounds of division before committing to terminal differentiation, fusing with the host fibers or generating new myofibers to reconstruct damaged tissue (Charge and Rudnicki, 2004). In several diseases and conditions affecting muscle, a reduction in muscle mass is seen that is associated with reduced numbers of satellite cells and a reduced ability of the satellite cells to repair, regenerate and grow skeletal muscle. A few exemplary diseases and conditions affecting muscle include wasting diseases, such as cachexia, muscular attenuation or atrophy, including sarcopenia, ICU-induced weakness, surgery-induced weakness (e.g., following knee or hip replacement), and muscle degenerative diseases, such as muscular dystrophies. The process of muscle regeneration involves considerable remodeling of extracellular matrix and, where extensive damage occurs, is incomplete. Fibroblasts within the muscle deposit scar tissue, which can impair muscle function, and is a significant part of the pathology of muscular dystrophies.
Muscular dystrophies are genetic diseases characterized by progressive weakness and degeneration of the skeletal or voluntary muscles which control movement. The muscles of the heart and some other involuntary muscles are also affected in some forms of muscular dystrophy. In many cases, the histological picture shows variation in fiber size, muscle cell necrosis and regeneration, and often proliferation of connective and adipose tissue. The progressive muscular dystrophies include at least Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), Limb-Girdle muscular dystrophies, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), Myotonic dystrophy (Steinert's disease) and congenital muscular dystrophies.
Currently there is no cure for these diseases, but certain medications and therapies have been shown to be effective. For instance, corticosteroids have been shown to slow muscle destruction in Duchene muscular dystrophy patients. While corticosteroids can be effective in delaying progression of the disease in many patients, long-term corticosteroid use is undesirable due to unwanted side effects.
PCT Application No. WO 2004/113513 (Rudnicki et al.) discloses methods and compositions for modulating proliferation or lineage commitment of an atypical population of CD45+Sca1+ stem cells, located outside the satellite stem cell compartment, by modulating myogenic determination of Wnt proteins.
The Wnt family of genes encode over twenty cysteine-rich, secreted Wnt glycoproteins that act by binding to Frizzled (Fzd) receptors on target cells. Frizzled receptors are a family of G-protein coupled receptor proteins. Binding of different members of the Wnt-family to certain members of the Fzd family can initiate signaling by one of several distinct pathways. In the termed canonical pathway, activation of the signaling molecule, Disheveled, leads to the inactivation of glycogen synthase kinase-3 (GSK-3β), a cytoplasmic serine-threonine kinase. The GSK-3β target, β-catenin, is thereby stabilized and translocates to the nucleus where it activates TCF (T-cell-factor)-dependant transcription of specific promoters (Wodarz, 1998, Dierick, 1999). In the non-canonical, or planar cell polarity (PCP) pathway, binding of Wnt to Fzd also activates Disheveled, which in this case activates RhoA, a small g protein. Activation of the PCP pathway does not result in nuclear translocation of β-catenin.
Wnt signaling plays a key role in regulating developmental programs through embryonic development, and in regulating stem cell function in adult tissues (Clevers, 2006). Wnts have been demonstrated to be necessary for embryonic myogenic induction in the paraxial mesoderm (Borello et al., 2006; Chen et al., 2005; Tajbakhsh et al., 1998), as well in the control of differentiation during muscle fiber development (Anakwe et al., 2003). Recently, the Wnt planar cell polarity (PCP) pathway has been implicated in regulating elongation of differentiating myocytes in the developing myotome (Gros et al., 2009). In the adult, Wnt signaling is necessary for the myogenic commitment of adult CD45+/Sca1+ stem cells in muscle tissue following acute damage (Polesskaya et al., 2003; Torrente et al., 2004). Other studies suggest that canonical Wnt/β-catenin signaling regulates myogenic differentiation through activation and recruitment of reserve myoblasts. In addition, Wnt/β-catenin signaling in satellite cells within adult muscle appears to control myogenic lineage progression by limiting Notch signaling and thus promoting differentiation. Thus, traditionally, it has been assumed that Wnt proteins act as stem cell growth factors, promoting the proliferation and differentiation of stem cells and/or progenitor cells.
This has established a potential role for Wnts in the treatment of myodegenerative diseases. However, the poor protein solubility of Wnts has hindered their use in recombinant protein therapy. In addition, Wnt proteins are lipidated during posttranscriptional processing prior to secretion and contain a vast number of hydrophobic amino acid residues, leading to low water solubility and resultantly deterring systemic delivery. Thus, current strategies for delivering Wnts, such as Wnt7a, for example, limit the use of Wnt polypeptide therapies for treating myodegenerative diseases.
Accordingly, there is a need in the art for modified Wnt polypeptides having increased solubility and bioavailability to effectively treat myodegenerative diseases.